Identification of Proteins Important for Male Osteoporosis Osteoporosis is a major public health problem, mainly characterized by low bone mineral density (BMD). Variation of BMD is largely genetically determined (heritability of >60%). Some BMD genes/genomic regions are gender specific. Although women have lower BMD than men, men suffer significantly higher mortality rate upon osteoporotic fractures. However, studies on osteoporosis have largely been focused on women. Few genetic epidemiological studies and no proteomic epidemiological study on osteoporosis have been performed on men. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs) are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. Proteomics is a powerful state-of-the-art strategy in genetic dissection of complex diseases, such as osteoporosis. However, a major problem affecting the power of current most proteomic studies is the limited detection of low abundance proteins and proteins with extreme isoelectric point, molecular weight, and hydrophobicity, especially membrane proteins. A NOVEL approach to this problem is subcellular proteome extraction to stepwise isolate proteins from membrane, cytosol, nucleus, and cytoskeleton E fractions followed by sensitive 2D-nanoLC-ESI-MS/MS for fractioning and identifying significant proteins. Our hypothesis is that changes in the protein expression profiles in BMMSCs and PBMs underlie molecular mechanisms of BMD variation and are associated with osteoporosis in men. Our major goals here are to identify proteins differentially expressed in BMMSCs and PBMs in men with high vs. low BMD and thus identify proteins (and their genes) associated with male osteoporosis in BMMSCs and PBMs. We will recruit 120 otherwise healthy Caucasian males at peak bone mass ages of 25-50, including 60 subjects with low and 60 with high BMD (age matched population bottom or top 20% respectively in terms of BMD). Half of the sample (30 low vs. 30 high BMD subjects) will serve as the "discovery cohort" and the other half (30 low vs. 30 high BMD subjects) will serve as the "replication cohort". We will take fresh bone marrow and peripheral blood samples from each male subject, as we do in our ongoing NIH SCOR projects for female subjects. BMMSCs and PBMs will be isolated by subcellular proteome extraction for membrane proteins together with proteins of cytosolic, nuclear, and cytoskeletal fractions. Proteomic profiling experiments and analyses will be performed on the isolated protein samples from the discovery E cohort using 2D-nanoLC-ESI-MS/MS . Significant differentially expressed proteins identified will be verified by Western blotting using samples from the replication cohort. The major results (particularly those obtained from PBMs) of this study may be used to design customary diagnostic protein antibody chips and/or protein markers for prognosis of male osteoporosis. In combination with our ongoing projects for identifying risk genes of osteoporosis through genome-wide DNA association scan and genome-wide mRNA expression study of osteogenic cells, this study will powerfully and efficiently identify genes and some of their functions for male osteoporosis. PUBLIC HEALTH RELEVANCE: Osteoporosis is a disease with severe bone loss and a significantly increased risk of low trauma fractures. Male osteoporosis, compared with female osteoporosis, has higher mortality rate upon fractures. This project aims to identify proteins that are important to male osteoporosis. The findings will provide essential scientific basis for effective prevention, diagnosis and treatment of the disease.